Congenital nerve sheath tumor in a dog

Abstract

A 5-wk-old, 10.6-kg, intact female Leonberger dog was presented for evaluation of a mass on the left ventrolateral thorax that had been present since birth. A biopsy of the mass revealed an invasive, unencapsulated spindle-cell population arranged in bundles and concentric whorls (pseudo-onion bulb formations) with multifocal melanocytic differentiation. Neoplastic cells in pseudo-onion bulbs immunolabeled strongly for glial acidic fibrillary protein and PGP9.5 and moderately for S100 and Sox10. The supporting matrix had strong immunolabeling for laminin. Cells had multifocal immunolabeling for NeuN, melan A, and PNL2. Collectively, these histopathologic characteristics support a diagnosis of congenital nerve sheath tumor, which is rarely described in dogs.

Nerve sheath tumors (NSTs) arise from the cells that surround and support peripheral nerves, including Schwann cells, perineurial cells, and fibroblasts, and can be benign or malignant. Benign NSTs generally carry a good prognosis; malignant NSTs (MNSTs) can invade local connective tissues and muscle and are often difficult to resect.^4,10,30 NSTs have been reported in adult dogs, cattle, horses, goats, sheep, and cats.^{2,7,25,27,28,29} The most common locations of NSTs in adult dogs are the roots of spinal nerves, but they have also been described in the liver, eye, eyelid, spleen, adrenal gland, diaphragm, lung, urinary bladder, tongue, and intestine. ³² Congenital NSTs are an uncommon neoplasm in pediatric patients and are rarely described in the veterinary literature. ¹⁶

A 5-wk-old, 10.6-kg, intact female Leonberger dog was presented to the Colorado State University–Veterinary Teaching Hospital (CSU-VTH; Fort Collins, CO, USA) because of a mass on the left ventrolateral thorax that had been present since birth. It was initially presumed to be a histiocytoma, and treatment was not pursued. However, the mass continued to grow and became ulcerated because of chronic irritation.

Upon presentation at the CSU-VTH, the 4.6 × 5.8 × 2.7-cm, soft mass had irregular borders and was freely movable on the left ventrolateral thorax. The left axillary lymph node was aspirated for cytology and was read as a heterogeneous lymphoid population with mildly increased numbers of plasma cells, intermediate lymphocytes, neutrophils, and occasional vacuolated macrophages and eosinophils, consistent with a reactive lymph node with possible neutrophilic lymphadenitis. Standard 3-view thoracic radiographs confirmed a large, lobulated mass in the soft tissues of the cranial left ventrolateral thorax; no evidence of communication with the thoracic cavity or metastatic disease was noted. The mass was surgically excised when the patient was 8-wk-old and submitted for histologic analysis. The surgical sample was fixed for >24 h in 10% neutral-buffered formalin, processed routinely, embedded in paraffin, sectioned at 5 µm, and sections were stained with H&E.

Histologically, the mass was incompletely excised and had extensive peripheral invasion into the dermis, subcutis, and skeletal muscle (Fig. 1A, 1B). The mass consisted of an unencapsulated spindle-cell population arranged in bundles, concentric whorls (pseudo-onion bulb formations), and vague streams ( Fig. 1C ) with occasional melanocytic differentiation ( Fig. 1D ) supported by prominent microvasculature and a collagenous-to-myxoid stroma ( Fig. 1E ). Neoplastic cells had indistinct borders, scant eosinophilic cytoplasm, and oval nuclei with finely stippled chromatin. Cytoplasmic brown pigment in cells was confirmed as melanin with a Fontana Masson preparation ( Fig. 1F ). Alcian blue stained weakly in areas of myxoid stroma ( Fig. 1G ). Anisocytosis and anisokaryosis were mild to moderate, and there were 9 mitoses in 10 hpf (400×; equivalent to 2.37 mm²). Mature mast cells were scattered within the mass.

Figure 1.

Congenital nerve sheath tumor in a dog. A. Mass is highly infiltrative (asterisks). H&E. B. Invasion of mass into skeletal muscle. H&E. C. Neoplasm with both perineurial differentiation (* = pseudo-onion bulb formations) and neurofibromatous differentiation with “shredded carrot” collagen (arrows). H&E. D. Occasional cells have intracytoplasmic brown-to-black granular pigment (melanin). H&E. E. Some regions have a reticular or collagenous-to-myxoid stroma. Mitotic figure in the lower right of the image. H&E. F. Melanocytic differentiation confirmed with Fontana Masson stain. G. Myxomatous matrix confirmed with alcian blue stain.

Immunohistochemical staining was performed for glial fibrillary acidic protein (GFAP), S100, protein gene product 9.5 (PGP9.5), Sox10, laminin, neuronal nuclear antigen (NeuN), melan A, PNL2, periaxin, CNPase, neurofilament protein (NFP), vimentin, and Ki67 ( Table 1 ). Central fibers of pseudo-onion bulb formations immunolabeled strongly for GFAP, moderately for S100, and strongly for PGP9.5; scattered individual nuclei immunolabeled for Sox10 ( Fig. 2A–D ). Strong immunolabeling for laminin in the matrix supported pseudo-onion bulbs ( Fig. 2E ). Sox10 immunolabeling was strong in melanocytic cells scattered throughout the mass. Nuclear NeuN immunolabeling was near-diffuse throughout the mass ( Fig. 2F ). Pigmented neoplastic cells and moderate numbers of non-pigmented neoplastic cells had strong cytoplasmic immunolabeling for melan A and PNL2 (Fig. 2G, 2H). Diffuse cytoplasmic vimentin immunolabeling was also strong (not shown). The Ki67 proliferative index was 9.7% (number of immunolabeled cells per 500 manually counted tumor cells). No immunolabeling was found for periaxin, NFP, or CNPase.

Table 1.

Details of antibodies and methods used for immunohistochemistry investigation of a congenital nerve sheath tumor in a dog.

Antibody	Host	Source	Clone	Antigen retrieval*	Dilution	Chromogen	Autostainer or manual
Ki67	Mouse monoclonal	Leica, PA0230	K2	ER2-20	RTU	AP Mouse Red	Leica BOND III
GFAP	Mouse monoclonal	Leica, PA0026	GA5	ER2-20	RTU	AP Mouse Red	Leica BOND III
S100	Rabbit polyclonal	DAKO, IR504	Polyclonal	ER2-20	RTU	RAB Red	Leica BOND III
PGP9.5	Rabbit polyclonal	Abcam, AB15503	N/A	ER1, 20 min	1:500	DAB	Leica BOND MAX
Sox10	Mouse monoclonal	Abcam, AB212843	SOX10 991	Steam citrate	1:200	Vector Nova Red	Manual
Laminin	Rabbit polyclonal	BioGenex, PU078-UP	N/A	Protease	4:200	Vector Nova Red	Manual
NeuN	Mouse monoclonal	Millipore Sigma, MAB377	A60	Protease	0.5:200	Vector Nova Red	Leica BOND III
Melan A	Mouse monoclonal	Leica, PA0233	A103	ER2-20	RTU	AP Mouse Red	Leica BOND III
PNL2	Mouse monoclonal	Cell Marque, 365M-96	PLN2	ER2-20	Con	AP Mouse Red	Leica BOND III
Periaxin	Rabbit polyclonal	Millipore Sigma, NPA001868–100 µL	N/A	Steam citrate	0.2:200	Vector Nova Red	Manual
CNPase	Mouse monoclonal	Abcam, Ab6319	11-5B	ER2-20	Con	AP Mouse Red	Leica BOND III
NFP	Mouse monoclonal	DAKO, M0762	2F11	ER2-20	1:150	AP Mouse Red	Leica BOND III
Vimentin	Mouse, monoclonal	Leica, PA0640	V9	ER2-20	RTU	AP Mouse Red	Leica BOND III

Con = concentrate; GFAP = glial fibrillary acidic protein; NFP = neurofilament protein; RTU = ready to use.

^* Leica BOND Epitope Retrieval Solution 2 (ER2) is an EDTA-based heat-induced epitope retrieval (HIER) solution, pH 9; all sections underwent 20 min of HIER.

Figure 2.

Immunohistochemistry (IHC) of a congenital nerve sheath tumor in a dog. A. Strong cytoplasmic GFAP immunolabeling in central fibers of pseudo-onion bulb formations. GFAP IHC. B. Moderate cytoplasmic S100 immunolabeling in central fibers of pseudo-onion bulb formations. S100 IHC. C. Strong PGP9.5 immunolabeling in central fibers of pseudo-onion bulb formations. PGP9.5 IHC. D. Strong nuclear Sox10 immunolabeling in individual cells within pseudo-onion bulb formations. Sox10 IHC. E. Strong laminin immunolabeling in central fibers of pseudo-onion bulb formations. Endothelial basement membranes are also immunolabeled. Laminin IHC. F. Most cells have strong nuclear NeuN immunolabeling. NeuN IHC. G. Strong cytoplasmic melan A immunolabeling in scattered neoplastic cells. Melan A IHC. H. Strong cytoplasmic PLN2 immunolabeling in pigmented and scattered non-pigmented cells. PLN2 IHC.

NSTs can arise from Schwann cells, from fibroblasts in the epineurium or endoneurium, or from perineurial cells that form the perineurium. ¹⁸ Benign NSTs are subclassified as neurofibroma (combination of Schwann cells and epi- or endoneurial fibroblasts), schwannoma (Schwann cells), perineurioma (perineurial cells), and hybrid tumors, all of which may arise directly from a nerve or be found in other soft tissues. ¹⁸ MNSTs may share some of these features and are classified histologically as conventional (with a distinctive spindle-cell morphology), divergent (featuring bone, cartilage, and/or muscle), perineurial, and epithelioid MNSTs.^3,21,32

The findings in our case are consistent with a diagnosis of congenital NST with hybrid perineurial and neurofibromatous differentiation. The pseudo-onion bulb formations are a distinctive perineurial morphology. Occasional regions featured a reticular-to-myxoid pattern (Fig. 1E), which is also reported in perineuriomas in people. ²⁰ The collagenous component sometimes had a wavy contour reminiscent of the “shredded carrot” appearance of neurofibromas (Fig. 1C). Neurofibromas consist of a mixture of Schwann cells, axons, and fibroblasts, and they commonly include mast cells. ³ Pigmented neurofibromas and schwannomas (malignant melanotic NST) that label with melanocytic markers are both reported in the human literature; the combined presence of mast cells and cells with melanocytic differentiation further supports a hybrid differentiation.^9,12 Nuclei in these areas were much more plump and cells were more mitotically active than the typical ovoid, hyperchromatic nuclei found in classical neurofibromas. ³⁴ The high degree of peripheral invasion into soft tissues also raised concern for potential malignancy at the time of diagnosis. However, the overall lack of pleomorphism, lack of nuclear atypia, and absence of necrosis were features inconsistent with malignancy. ¹⁹ The owner reported no signs of recurrence 14 mo postoperatively, which also suggests a clinically benign neoplasm.

Definitive diagnosis of NSTs in veterinary medicine can be challenging because of the significant overlap in histologic features among various subtypes and with soft-tissue sarcomas. The prominent perineurial differentiation aided the diagnosis in our case, which was further supported by immunohistochemical findings. Pseudo-onion bulb formations consist of concentric layers of perineurial cells surrounding a central Schwann cell and axon. In our case, central portions of pseudo-onion bulbs immunolabeled strongly for GFAP and PGP9.5 and moderately for S100, with scattered nuclear Sox10 immunolabeling typical of Schwann cell origin (Fig. 2A–D). Pseudo-onion bulb structures were supported by laminin, a Schwann cell product that contributes to the basement membranes of peripheral nerves (Fig. 2E). The surrounding presumptive perineurial cells had no immunolabeling for these markers, similar to a case series of canine intraneural perineuriomas, but expressed NeuN and variable melan A and PNL2, corroborating neuroectodermal origin (Fig. 2F–H). ³⁵ Immunohistochemical staining profiles for canine NSTs vary among studies, likely reflecting the varied histogenesis or differentiation of these tumors and selection or availability of different immunohistochemical panels.^5,11,32,35 An interesting feature in our case was the finding of pigmented and non-pigmented cells of melanocytic origin along with the pseudo-onion bulb formations. Both the human and veterinary literature have reported pigmented NST variants, which are hypothesized to result from the shared embryonic neural crest origin of melanocytes and Schwann cells.^13,24,33

In dogs, most benign and malignant NSTs arise from the spinal nerves of adult animals.^10,14,18 MNSTs are locally invasive and slow to metastasize. Prognosis may be poor depending on their location, especially those invading the spinal canal or involving cranial nerves. ¹⁰ Our case was a congenital NST arising in the skin, which to our knowledge has been reported only once previously in a dog. In a search of Google, PubMed, CAB Direct, Web of Science, and Scopus, using the search terms “congenital nerve sheath tumor”, “congenital neurofibroma”, “congenital perineurioma”, “congenital schwannoma”, “canine”, and “dog”, we retrieved one case of congenital neurofibroma in a 7-wk-old puppy that lacked histologic and immunohistochemical characterization. ¹⁶

To date, congenital cases of NSTs have only been detailed in humans, where they are typically associated with neurofibromatosis 1 (NF1).^22,26 NF1 is an autosomal dominant disorder caused by mutations in the NF1 gene, which encodes the tumor suppressor protein neurofibromin. This gene has a notoriously stochastic mutation rate in the human genome because of its large size. A combination of random and inherited mutations leads to 1 in every 3,000 live births developing the condition. ¹⁵ The NF1 gene is expressed in many cell types, including those of neural crest origin, such as glial cells, Schwann cells, and melanocytes early in development. ³¹ Thus, individuals with NF1 are more likely to develop both benign and malignant neoplasms of the peripheral and central nervous systems. The hallmark neoplasm associated with NF1 is the neurofibroma, which is categorized as either a cutaneous or plexiform subtype. Cutaneous neurofibromas typically develop near puberty and are benign nodular neoplasms within the dermis. Plexiform neurofibromas (pNF) are usually congenital, are located around larger peripheral nerves, and often prove challenging to manage because of their extensive invasion. Other tumors, including MNST and glioma also occur at increased frequency, with half of all MNSTs occurring in patients with NF1. ³¹

NF-like syndromes have been reported in cattle and dogs. NSTs are one of the most common neoplasms identified at slaughter in cattle.^6,14 Typically, NSTs in cattle are non-cutaneous. However, reports of multiple cutaneous neurofibromas in Holsteins support a hereditary component, similar to NF1 in people. In addition, one case of subcutaneous melanotic neurofibroma has been reported in a steer.^14,17 NF-like syndromes have also been proposed in dogs, although a genetic component has not been established.^8,16 Interestingly, multiple congenital cutaneous neurofibromas and a meningeal rhabdomyosarcoma were reported in a 7-wk-old Labrador Retriever. ¹⁶ Although animals do not precisely recapitulate the genetic and morphologic features of NF in people, spontaneous NF-like disease in cattle, pigs, and dogs may serve as a useful preclinical translational model. ²³

Based on limited prognostic information and the concern for invasive behavior, options offered to the client in our case included radical surgery or radiation therapy to treat any potential recurrence. The client declined further treatment options. Given the lack of veterinary literature on congenital NSTs, no consensus exists on the optimal treatment for animals. Benign childhood NSTs are reported to have an excellent prognosis, although a small percentage of pNF in NF1 patients will undergo malignant transformation to MNST. ³¹ Childhood MNST, on the other hand, carries a poor prognosis with a high incidence of local recurrence, high metastatic rate, and increased mortality. ¹ The patient in our case was healthy with no signs of local recurrence 14 mo postoperatively. Prognosis is uncertain given the lack of veterinary literature.